The invention is related to the detection of the transduction of an extracellular signal by an intracellular signal transduction pathway. In particular, the invention relates to methods and compositions useful for identifying a test compound as an agonist or antagonist of a cellular receptor.
The identification of biological activity in new molecules has historically been accomplished through the use of in vitro assays or whole animals. Intact biological entities, either cells or whole organisms, have been used to screen for anti-bacterial, anti-fungal, anti-parasitic and anti-viral agents in vitro. Cultured mammalian cells have also been used in screens designed to detect potential therapeutic compounds. A variety of bioassay endpoints have been exploited in cell screens including the stimulation of growth or differentiation of cells, changes in cell motility, the production of particular metabolites, the expression of specific proteins within cells, altered protein function, and altered conductance properties. Cytotoxic compounds used in cancer chemotherapy have been identified through their ability to inhibit the growth of tumor cells in vitro and in vivo. In addition to cultures of dispersed cells, whole tissues have served in bioassays, as in those based on the contractility of muscle.
In vitro testing is a preferred methodology in that it permits the design of high-throughput screens: small quantities of large numbers of compounds can be tested in a short period of time and at low expense. Optimally, animals are reserved for the latter stages of compound evaluation and are not used in the discovery phase, inasmuch as the use of whole animals is labor-intensive and extremely expensive.
The search for agonists and antagonists of cellular receptors has been an intense area of research aimed at drug discovery because of the elegant specificity of these molecular targets. Drug screening has been carried out using whole cells expressing functional receptors and, recently, binding assays employing membrane fractions or purified receptors have been designed to screen compound libraries for competitive ligands.
G protein-coupled receptors (GPCRs) are a particularly important category of cell surface receptors. The medical importance of these receptors is evidenced by the fact that more than 60% of all commercially available prescription drugs work by interacting with known GPCRs. Hundreds, if not thousands, of receptors convey messages through heterotrimeric G proteins, of which at least 17 distinct forms have been isolated. Most G protein-coupled receptors are comprised of a single protein chain that is threaded through the plasma membrane seven times. Such receptors are often referred to as seven-transmembrane receptors (STRs). More than a hundred different GPCRs have been found, including many distinct receptors that bind the same ligand, and there are likely many more GPCRs awaiting discovery. The development of new drug discovery assays to identify novel modulators of GPCRs would be of tremendous benefit.
The heterologous expression of recombinant mammalian G protein-coupled receptors in mammalian cells which do not normally express those receptors has been described as a means of studying receptor function for the purpose of identifying agonists and antagonists of those receptors. For example, the human muscarinic receptor (HM1) has been functionally expressed in mouse cells (Harpold et al. U.S. Pat. No. 5,401,629). The rat V1b vasopressin receptor has been found to stimulate phosphotidylinositol hydrolysis and intracellular Ca2+ mobilization in Chinese hamster ovary cells upon agonist stimulation (Lolait et al. (1995) Proc. Natl. Acad Sci. USA 92:6783-6787). These types of ectopic expression studies have enabled researchers to study receptor signaling mechanisms and to perform mutagenesis studies which have been useful in identifying portions of receptors that are critical for ligand binding or signal transduction.
Experiments have also been undertaken to express functional G protein-coupled receptors in yeast cells. For example, U.S. Pat. No. 5,482,835 to King et al. describes a transformed yeast cell which is incapable of producing a yeast G protein xcex1 subunit, but which has been engineered to produce both a mammalian G protein xcex1-subunit and a mammalian receptor which is xe2x80x9ccoupled toxe2x80x9d (i.e., interacts with) the aforementioned mammalian G protein xcex1-subunit. Specifically, U.S. Pat. No. 5,482,835 reports expression of the human beta-2 adrenergic receptor (xcex22AR), a seven transmembrane receptor (STR), in yeast, under control of the GAL1 promoter, with the xcex22AR gene modified by replacing the first 63 base pairs of coding sequence with 11 base pairs of noncoding and 42 base pairs of coding sequence from the STE2 gene. (STE2 encodes the yeast xcex1-factor receptor.) It was found that the modified xcex22AR was functionally integrated into the membrane, as shown by studies of the ability of isolated membranes to interact properly with various known agonists and antagonists of xcex22AR. The ligand binding affinity for yeast-expressed xcex22AR was said to be nearly identical to that observed for naturally produced xcex22AR.
U.S. Pat. No. 5,482,835 also describes co-expression of a rat G protein xcex1-subunit in yeast strain 8C, which lacks the cognate yeast protein. Ligand binding resulted in G protein-mediated signal transduction. U.S. Pat. No. 5,482,835 further teaches that these cells may be used in screening compounds for the ability to affect the rate of dissociation of Gxcex1 from Gxcex2xcex3 in a cell. For this purpose, the cell further contains a pheromone-responsive promoter (e.g., BAR1 or FUS1), linked to an indicator gene (e.g. HIS3 or lacZ). The cells are placed in multi-titer plates, and different compounds are placed in each well. The colonies are then scored for expression of the indicator gene. DNA vectors and host yeast cells for use in the method are also disclosed (see U.S. Pat. No. 5,739,029).
U.S. Pat. No. 5,789,184 describes yeast cells engineered to express a heterologous kinase as a yeast pheromone system protein surrogate, and a heterologous polypeptide. The yeast cells are used in assays to screen for peptides that modulate the activity of non-yeast surrogates.
U.S. Pat. No. 5,879,591 describes yeast cells engineered to express a heterologous protein (e.g., a farnesyl transferease) which functions as a surrogate for, and performs the function of, a yeast pheromone system protein, as well as a heterologous polypeptide. The yeast cells are useful in screening assays to identify polypeptides which modulate the interaction of the surrogate with the yeast pheromone system.
U.S. Ser. No. 08/322,137 describes yeast cells engineered to express both a surrogate, e.g. a G protein-coupled receptor, of a pheromone pathway component and a potential peptide modulator of the surrogate. This is performed in such a manner that inhibition or antagonism of the surrogate by the peptide modulator affects a screenable or selectable trait of the yeast cell. Also included are mechanisms by which the signal-to-noise ratio of the system may be improved. The yeast cells are useful in assays to screen for peptides that modulate the activity of endogenous and heterologous yeast pheromone system surrogates.
Published PCT international application WO 98/13513 describes methods for identifying modulators of heterologous receptors expressed in yeast. Modulators are identified by detecting an alteration in a signal produced by an endogenous yeast signaling pathway.
Published PCT international application WO 99/18211 describes novel yeast cells which express a heterologous G protein coupled receptor and mutant and/or chimeric G protein subunit molecules which serve to functionally integrate the heterologous receptor into the pheromone signaling pathway of the yeast cell.
It is an object of the present invention to provide a novel, rapid, reproducible, robust assay system for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular receptor or ion channel of a cell. More particularly, the invention provides a highly sensitive assay system for the identification of agonist or antagonist activity of a test compound for a specific receptor.
Thus, in one aspect, the invention provides a method for identifying a test compound that modulates a heterologous receptor in a cell. The method comprises providing a first cell containing a heterologous receptor that is functionally integrated into a first signal transduction pathway of this first cell, where a signal molecule is produced by the first cell upon activation of the first signal transduction pathway; and providing a second cell which is responsive to production of the signal molecule by the first cell, such that a detectable signal is generated by the second cell in response to production of the signal molecule by the first cell. Upon contacting the first cell with the test compound, modulation of the heterologous receptor by the test compound is indicated by generation of the detectable signal by the second cell.
For example, a first cell, termed the xe2x80x98testxe2x80x99 cell, containing a desired receptor (either native or heterologous) which is functionally coupled to a first signal transduction pathway, is challenged with a test compound. If the test compound has a stimulatory (agonist) effect upon the receptor, a signal will be transmitted through the coupled signal transduction pathway; resulting in production (e.g., secretion) of a signal molecule by the test cell. The signal compound stimulates a second xe2x80x9cdetectorxe2x80x9d cell. The detector cell is responsive to (e.g., by expressing receptors for) the signal molecule. In one embodiment, the detector cell expresses a receptor for the signal molecule such that upon binding of the signal molecule, a second signal is transmitted through a second pathway functionally coupled to this second receptor. Stimulation of this second pathway in the detector cell can be measured either by some factor intrinsic to this second pathway, or more conveniently, by the inclusion of a reporter gene coupled to the pathway. Thus, signaling through the pathway stimulates the expression of a reporter molecule which confers a detectable phenotype upon the detector cell (such as a colorimetric, luminescent, or growth phenotype). The test and detector cells may be in mixed culture, may be separated by a semipermeable membrane, or may be entirely separate, with only the extracellular material from the test cell culture contacted with the detector cells.
In a preferred embodiment, the present invention provides a novel and sensitive alpha factor readout assay to assess the signal transduction activity through the pheromone response pathway in yeast cells. In this embodiment, a MATxcex1 yeast test cell, containing a heterologous G-protein coupled receptor (GPCR), is stimulated with a ligand of the GPCR. If reactive with the ligand, this stimulation results in increased xcex1-factor expression and secretion; the amount of xcex1-factor secretion is indicative of the activity of the pheromone signaling pathway in the test cell. The secreted xcex1-factor binds to an xcex1-factor receptor on a second, detector yeast cell of MATa mating type. Upon binding of xcex1-factor, the pheromone response pathway in this detector cell is activated. The detector cell may also include an indicator gene (such as lacZ) operatively linked to a promoter sensitive to upregulation of this second pathway (such as FUS1), such that the readout from the indicator gene yields a determination of the quantity of xcex1-factor present. Alternatively, a growth readout may be used, wherein expression of the indicator gene (such as LEU2) corrects for an auxotrophy and permits growth of the detector cells on selective medium.
The invention also provides methods by which the sensitivity of the assay may be increased. In one embodiment, for example, the invention provides for the elimination of constitutive background expression and secretion of xcex1-factor in an S. cerevisiae test cell by inactivation of the MFxcex11 gene in order to improve the detection limits of the assay. In other embodiments, the detector cell also comprises one or more gene mutations, the effect of which is to increase significantly the sensitivity of the response of the second pathway to receptor-dependent activation.
In another embodiment, the invention provides a method for identifying a compound that modulates production of a gene product produced by a cell. The method comprises providing a first cell, the first cell containing a surrogate of a signal transduction pathway of the first cell, where a gene product is produced by the surrogate upon activation of the signal transduction pathway, and also providing a second cell having means for generating a detectable signal and being responsive to the gene product, such that a detectable signal is generated by the second cell in response to stimulation of the second cell by the gene product, and contacting the first cell with a test compound; whereby the modulation is indicated by generation of the detectable signal by the second cell.
In another aspect, the invention is directed to a method for detecting a gene product produced by a cell. The method comprises causing a signal to be transmitted through a signal transduction pathway of a first cell, such that a gene product is produced by the first cell upon transmission of the signal through the signal transduction pathway; and detecting the gene product by means of a second cell which is responsive to the gene product produced by the first cell, where the second cell generates a detectable signal in response to production of the gene product by the first cell. In a preferred embodiment, the method further comprises providing a first cell, this first cell containing a surrogate of a signal transduction pathway of the first cell, where a gene product is produced by the surrogate upon activation of the signal transduction pathway; and also providing a second cell, this second cell having means for generating a detectable signal, such that a detectable signal is generated by the second cell in response to stimulation of the second cell by the gene product. In a particularly preferred embodiment, the signal transduction pathway is a yeast pheromone response pathway.
Yet another aspect of the invention is directed to recombinant cells. In one embodiment, the invention provides a recombinant cell comprising a surrogate of a signal transduction pathway of said cell, where a signal molecule is produced by the surrogate upon activation of the signal transduction pathway, and where production of the signal molecule is capable of detection by a second cell having means for generating a detectable signal, such that a detectable signal is generated by the second cell in response to stimulation of the second cell by the signal molecule. In a preferred embodiment, the signal transduction pathway is a yeast pheromone response pathway. In another preferred embodiment, the signal molecule is a gene product, with a natural product being particularly preferred.
In another embodiment, the invention provides a mixture of recombinant cells comprising a first recombinant cell and a second recombinant cell, where the first cell contains a surrogate of a signal transduction pathway of the cell, such that a signal molecule is produced by the surrogate upon activation of the signal transduction pathway, and where the second cell contains means for generating a detectable signal such that a detectable signal is generated by the second cell in response to stimulation of the second cell by the signal molecule.
In yet another embodiment, the invention provides a mixture of recombinant cells comprising a first recombinant cell and a second recombinant cell, where the first cell contains a heterologous receptor that is functionally integrated into a signal transduction pathway of the first cell, and produces a signal molecule upon activation of this signal transduction pathway, and where the second cell contains means for generating a detectable signal, and is responsive to the signal molecule produced by the first cell, such that a detectable signal is generated by the second cell in response to production of the signal molecule by the first cell.
In another aspect, the present invention provides a kit for screening of test compounds that modulate a heterologous receptor in a cell. This kit comprises a first cell having a heterologous receptor that is functionally integrated into a first signal transduction pathway of this first cell; a signal molecule is produced by this first cell upon activation of the first signal transduction pathway. This kit also comprises a second cell which is responsive to production of the signal molecule by the first cell, such that a detectable signal is generated by the second cell in response to production of the signal molecule by the first cell.